REGULATION OF INTERSTITIAL CELLS BY HORMONES AND NEUROPEPTIDES: MECHANISMS AND IMPLICATIONS FOR GASTROINTESTINAL MOTILITY
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Authors
Bartlett, Allison Marie
Issue Date
2025
Type
Dissertation
Language
en_US
Keywords
Enteric Nervous System , Gastrointestinal Hormones , Gastrointestinal motility , Interstitial cells of Cajal , PDGFR-alpha cells
Alternative Title
Abstract
ABSTRACT This dissertation investigates the roles and mechanisms of gastrointestinal (GI) hormones and peptides on the myogenic component of intestinal motility. The GI tract is a diverse and unique system that is exposed to a diverse external environment through the lumen. Every meal ingested is a new opportunity for this system to perform a unique set of actions, churning and breaking down solid substances into nutrients and filtering out unwanted or unnecessary components. To achieve this, the GI tract must orchestrate a delicate and highly regulated series of events involving chemical signaling elements of digestion and coordinated smooth muscle events. The myogenic component is composed of smooth muscle cells (SMC), Interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor alpha-positive (PDGFRα+) cells, called the “SIP” syncytium. ICC and PDGFRα+ cells are morphologically distinct interstitial cells that form networks in the circular and longitudinal muscle layers of the tunica muscularis. ICC and PDGFRα+ cells are closely associated with varicose processes of enteric motor neurons and form close networks surrounding the myenteric (Auerbach’s plexus). These networks of cells are electrically coupled through gap junctions and work together to drive precise and coordinated relaxation and contraction of smooth muscle underlying bigger motility motifs, like peristalsis.
It has been well established that GI hormones and peptides influence GI motility. However, research on the effects of GI hormones and peptides on interstitial cells is lacking. Research on this topic could provide a significant bridge to understanding GI motility disorders and diseases that are associated with GI dysmotility. Secretin, Glucagon-like peptide-2 (GLP-2), and calcitonin gene-related peptide (CGRP) are GI peptides that bind to G-protein coupled receptors (GPCRs) present on ICC and PDGFRα+ cells. The first aim in these studies was to establish a functional connection between peptide binding and interstitial cells. All peptides significantly inhibited the force of amplitude of contractions in muscle strip preparations in the small intestine for secretin, and the colon for GLP-2 and CGRP. Additionally, using mice expressing genetically encoded Ca²⁺ indicators (PDGFRα-iCre-GCaMP6f or Kit-iCre-GCaMP6f), we observed significant, cell- and tissue-specific changes in intracellular Ca²⁺ transients and waves in interstitial cells in the presence of the neurotoxin tetrodotoxin (TTX), which blocks neural action potentials and therefore disconnects smooth muscle responses from the enteric nervous system.
In murine and monkey small intestine muscle strip assays, secretin (100 nM) significantly reduced total contractile activity (AUC) and amplitude without affecting contraction frequency. In ex vivo GCaMP imaging, secretin decreased stochastic Ca2+ transients in ICC-DMP, both with and without TTX, while ICC-MY Ca2+ waves were unaffected. Similar reductions in AUC and amplitude, with unchanged frequency, were observed following cAMP elevation via forskolin or 8-Bromo-cAMP (cAMP-analogue). Ca2+ imaging confirmed reduced ICC-DMP activity under these conditions, with no effect on ICC-MY. Using CAMPER-Kit-iCre mice, where the FRET cAMP sensor is exclusively expressed in ICC. Secretin increased FRET signals, confirming cAMP elevation in ICC-DMP. Preincubation with PKA inhibitors predominantly attenuated secretin’s effects, whereas the EPAC inhibitor had partial effects. indicating that secretin actions are mainly mediated via the cAMP–PKA pathway.
Inositol 1,4,5-trisphosphate receptors (IP3Rs) on the endoplasmic reticulum are critical for the spontaneous, stochastic release of Ca2+ in ICC-DMP. To assess this mechanism in secretin responses, we used caged IP3 in ex vivo small intestine preparations from Kit-iCre-GCaMP6f mice. UV uncaging of IP3 during confocal imaging significantly increased Ca2+ transients in control ICC-DMP cells. This response was attenuated in the presence of secretin, indicating that secretin suppresses IP3R-mediated Ca²⁺ release.
Experiments investigating GLP-2 were focused on in the colon due to previously published RNA sequencing data showing the expression of the Glp2r in the murine colon ICC. Functional data showed that GLP-2 at 5 nM and 100 nM significantly inhibited total AUC, amplitude, and contractile events in proximal colon monkey muscle strips with and without the preincubation of TTX, as well as in mouse proximal colon. Confocal Ca2+ imaging of ICC-IM and ICC-MY in the proximal colon showed significant decreases in frequency, area, and spatial spread of Ca2+ events which was also visualized in isolated single cell ICC. The GLP2R is linked to the GPCR-Gαs and therefore as was seen in secretin experiments, we wanted to determine if cAMP was increased in ICC after the activation of the GLP2R. CAMPER-Kit-iCre mouse experiments in the proximal colon ICC-MY, confirmed with immunofluorescence colocalization, showed increases in cAMP levels after the addition of GLP-2 as well as in controls using forskolin.
PKA pathways were shown to crosstalk with PKG and subsequent activation of the IP3R protein regulator, Inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate (IRAG). PKG has been shown to phosphorylate IRAG at the ER membrane, negatively influencing the IP3R and preventing the release of Ca2+ from internal stores. Experiments investigating if this pathway is involved in the GLP-2 suppression of Ca2+ signaling in ICC was investigated. Using global IRAG (Mrvi1) knockout mice, the typical inhibitory effects of GLP-2 on colonic contractile events were not significantly affected. Also, In and ICC-specific Mrvi1-KO mice (Kit-iCre-Mrvi1-KO), GLP-2 did not as significantly reduce the total AUC as in controls. GLP-2 is a dynamic GI hormone that may influence ICC function via cAMP signaling as well as in IRAG activation to control Ca2+ handling in ICC and subsequent smooth muscle relaxation of the proximal colon.
The role of CGRPα in colonic motility remains underexplored. The CGRP receptor complex was found to be expressed in PDGFRα+ cells in the colon as seen in previously published RNA sequencing data from fluorescence activated cell sort (FACS) samples. Distal and proximal colon monkey and murine muscle contractions were inhibited in the presence of CGRPα with and without TTX. The CGRPR antagonist, olcegepant, blocked the effects of CGRPα. The small conductance Ca2+ activated potassium channel (K+) subtype 3, (SK3) is exclusively expressed in PDGFRα+ cells in the GI tract. Therefore, with the addition of the SK3 channel antagonist, apamin, the full inhibitory effect of CGRPα seen in controls was blocked in the distal colon and was partially blocked in the proximal colon. Electrical intracellular recordings from distal colon smooth muscles in the presence of TTX showed that CGRPα induced significant hyperpolarization of the resting membrane potential, which was reversed by apamin. In the PDGFRα+-SK3-KO mice, the inhibitory effects of CGRPα were not observed.
Ca2+ imaging of PDGFRα cells showed increased Ca²⁺ activity in PDGFRα-IM and PDGFRα-MY cells after CGRPα in the presence of TTX in both the distal and proximal colon. However, in Ca2+ imaging of ICC, CGRPα was shown to decrease Ca2+ events in proximal colon of ICC-IM and ICC-MY. RNAscope confirmed the expression of the receptor activity modifying protein 1 (RAMP1), which is an integral component that forms the CGRP receptor in PDGFRα cells in the colon. These results indicate that CGRPα bind to receptors on interstitial cells and ultimately leading to smooth muscle relaxation in the colon. This effect can be attributed in part to the activation of the SK3 channels in PDGFRα+ cells.
Collectively, the functional changes observed in ICC and PDGFRα⁺ cells in response to secretin, GLP-2, and CGRP represent significant findings and highlight promising avenues for future investigation.